The present invention relates to a temperature-controlled pH-dependant formation of ionic polysaccharide gels, such as chitosan/organo-phosphate aqueous systems, and methods of preparation thereof.
Chitosan is a commercially available inexpensive polymer, a derivative of chitin or poly(N-acetyl-glu-cosamine) materials. Chitosan is mainly composed of D-glucosamine units that are generated through catalyzed N-deacetylation of chitin, an insoluble biopolymer extracted from hard shells of marine living animals (fishes, crustaceans, shrimps, crabs . . . ) or synthesized by natural organisms (zygomycete, fungi . . . ). Chitosan is expected to have good viscoelastic properties, and has adequate tissue compatibility and biodegradability which renders it ideal for bioactive and resorbable implants. Poly-D-glucosamine chains are also known to potentially attach a large number of proteoglycan molecules and coexist with fibrous collagens to form aqueous gels. It is believed that the role of proteoglycans within the gel is to retain water and supply appropriate viscoelasticity. Resulting extracellular matrices are expected to offer compatible environments for cellular proliferation and tissue formation, especially for skin, ligament, bone and cartilage cells. As a consequence, chitosan attracts great interest for scaffolding or supporting materials of bioengineered artificial tissues.
Moreover, chitin and partially-acetylated chitosan derivatives have been extensively investigated for therapeutic substances or implantable materials. Biocompatibility of chitosan-based materials has been evaluated specifically for blood, wounds and bone. Immunological and genotoxic activities as well as stimulatory effects on macrophagic action have been also studied with various chitosan materials.
Chitosan and its derivatives has been widely explored for drug delivery system through gels (Ohya Y. et al. (1993) J. Micro-encapsulation, 10(1):1-9). Peptides delivery with chitosan was proposed to be effected nasally. Cationic colloidal drug carriers were proposed from chitosan-polycaprolactone systems. Wound healing and reconstructive devices made of chitosan materials have been proposed for open or corneal wounds, periodontal tissues and skin. Chitosan was specially evaluated in bone and dura matter and as an hemostatic.
Entrapment of living biologicals (cells, enzymes, etc . . . ) have been investigated with different chitosan products, however, in nearly all cases, living cells have been encapsulated within alginate/chitosan microbeads. Encapsulation of chondrocytes (cartilage cells) were proposed within calcium-alginate/chitosan beads.
Gelation of chitosan through polyphosphates has been promoted for encapsulating cells such as neural or musculo-squeletal tissues. Generally, chitosan in an acid/water medium was loaded with cell suspensions, and the resulting mixture was dropped in a buffered penta-sodium tri-polyphosphates so as to form cell-loaded chitosan beads and capsules. Entrapment of neural cells within polyphosphate-gelated chitosan beads has led to good cellular viability but low proliferation rate (Zielinski B. A. et al. (1994) Biomaterials, 15(13) :1049-1056). No large or specific three-dimensional shaped materials were proposed (Zielinski B. A. et al. (1994) Biomaterials, 15(13) :1049-1056). Polysaccharide capsules have been proposed for entrapping physiologically active cells such as Langerhans Islets (U.S. Pat. No. 4,391,909). Chitosan/hydrochloride cisplatin mixture were cross-linked and proposed as drug delivery systems.
Chitosan derivatives have been incorporated in numerous carrier composition or drug formulation. Chitosan materials such as wound filling materials or contraceptive products were also proposed (U.S. Pat. Nos. 4,956,350 and 4,474,769). Chitosan gels were again reported as supports for immobilizing and encapsulating living biomaterials such as cells, bacteria and fungi (U.S. Pat. No. 4,647,536). Ophthalmic drug delivery systems made of chitosan were also proposed for in situ gelating and forming (U.S. Pat. No. 5,422,116).
In U.S. Pat. No. 4,659,700, chitosan gels were prepared from glycerol/acid/water systems as biodegradable carriers for drug delivery. The resulting chitosan gels were reported to remain quite stable, keeping intact their three-dimensional shape for long periods and over a wide range of temperatures, particularly between 4 and 40xc2x0 C. Gels and gel-like materials were processed by dissolving 1.0 to 4.0% w/v chitosan within acid-water-glycerol solutions wherein acetic, formic or propionic acid and 10-90% glycerol proportions are used preferentially, and by neutralizing with liquid bases such the sodium, ammonium and potassium hydroxides or ammonia vapors. The pH of the resulting chitosan-glycerol gel materials is about pH 7.0. After neutralization, the resultant mixtures turn into gels upon standing, such gels resulting apparently from the interaction of chitosan, glycerol and water. No free glycerol or water were reported as being apparent. It must be noted, however, that such three-dimensionally shaped chitosan-glycerol gels will occur only when the solution is previously neutralized with a base. One-piece three-dimensional gels can be molded easily as well as gel-like membranes. The role of the glycerol component and chitosan-glycerol interactions is not elucidated.
In situ formed gels were also proposed with ionic polysaccharides in U.S. Pat. No. 5,587,175. A composition can be used as a medical device for drug delivery, the application of a diagnostic agent, or the prevention of post-operative adhesions, and is composed an aqueous liquid vehicle which is capable of being gelled in situ. It includes at least one ionic polysaccharide, at least one film forming polymer, and a medicament or pharmaceutical agent, water, and optionally, a counter-ion capable of gelating the ionic polysaccharide (U.S. Pat. No. 5,587,175). However, the gelation is reached by interaction between the ionic polysaccharide and the film-forming polymer, or by counter-ion induced cross-linking of the ionic polysaccharide. Other in situ forming gels are based upon polyoxyalkylene composition (U.S. Pat. No. 4,185,618) or polyoxyalkylene/polysaccharide mixture (U.S. Pat. No. 5,126,141) or alginate/cation mixture in situ (U.S. Pat. Nos. 4,185,618 and 5,266,326).
It would be highly desirable to be provided with a temperature-controlled pH-dependant formed polysaccharide gel which could be used to encapsulate cells and cellular material while retaining their biological activity.
It would be highly desirable to be provided with such a polysaccharide gel which would retain its solid or gel state at the physiological temperature or 37xc2x0 C.
One aim of the present invention is to provide a temperature-controlled pH-dependant formed polysaccharide gel which could be used to encapsulate cells and cellular material while retaining their biological activity.
Another aim of the present invention is to provide a polysaccharide gel which would retain its solid or gel state at the physiological temperature or 37xc2x0 C.
Another aim of the present invention is to provide a method for the preparation of such a polysaccharide gel.
In accordance with the present invention there is provided a polysaccharide based gel which comprises:
a) 0.1 to 5.0% by weight of chitosan or a chitosan derivative; and
b) 1.0 to 20% by weight of a salt of polyol or sugar selected from the group consisting of mono-phosphate dibasic salt, mono-sulfate salt and a mono-carboxylic acid salt of polyol or sugar;
wherein said gel is induced and stable within a temperature range from 20 to 70xc2x0 C. and is adapted to be formed and/or gelated in situ within a tissue, organ or cavities of an animal or a human.
The salt may be any of the following or in any of the following combination:
a) a mono-phosphate dibasic salt selected from the group consisting of glycerol, comprising glycerol-2-phosphate, sn-glycerol 3-phosphate and L-glycerol-3-phosphate salts;
b) a mono-phosphate dibasic salt and said polyol is selected from the group consisting of histidinol, acetol, diethylstilbestrol, indole-glycerol, sorbitol, ribitol, xylitol, arabinitol, erythritol, inositol, mannitol, glucitol and a mixture thereof;
c) a mono-phosphate dibasic salt and said sugar is selected from the group consisting of fructose, galactose, ribose, glucose, xylose, rhamnulose, sorbose, erythrulose, deoxy-ribose, ketose, mannose, arabinose, fuculose, fructopyranose, ketoglucose, sedoheptulose, trehalose, tagatose, sucrose, allose, threose, xylulose, hexose, methylthio-ribose, methylthio-deoxy-ribulose, and a mixture thereof;
d) a mono-phosphate dibasic salt and said polyol is selected from the group consisting of palmitoyl-glycerol, linoleoyl-glycerol, oleoyl-glycerol, arachidonoyl-glycerol, and a mixture thereof; and
e) glycerophosphate salt is a selected from the group consisting of glycerophosphate disodium, glycerophosphate dipotassium, glycerophosphate calcium, glycerophosphate barium and glycerophosphate strontium.
A preferred gel in accordance with one embodiment of the present invention is selected from the group consisting of chitosan-xcex2-glycerophosphate, chitosan-xcex1-glycerophosphate, chitosan-glucose-1-glycerophosphate, and chitosan-fructose-6-glycerophosphate.
The solid particulate or water-soluble additives may be incorporated within said polysaccharide gel prior to the gelation.
The drugs, polypeptides or non-living pharmaceutical agents may be incorporated within said polysaccharide gel prior to the gelation.
The living microorganisms, plant cells, animal cells or human cells may be encapsulated within said polysaccharide gel prior to the gelation.
The gel may be formed in situ sub-cutaneously, intra-peritoneally, intra-muscularly or within biological connective tissues, bone defects, fractures, articular cavities, body conduits or cavities, eye cul-de-sac, or solid tumors.
The gel of the present invention may be used as a carrier for delivering pharmaceutical agents in situ.
In accordance with the present invention there is also provided a method for producing a polysaccharide gel solution of the present invention, which comprises the steps of:
a) dissolving a chitosan or a chitosan derivative within an aqueous acidic solution of a pH from about 2.0 to about 5.0 to obtain an aqueous polysaccharide composition having a concentration of 0.1 to 5.0% by weight of a chitosan or of a chitosan derivative;
b) dissolving 1.0 to 20% by weight of a salt of polyol or sugar, wherein said salt is selected from the group consisting of mono-phosphate dibasic salt, mono-sulfate salt and a mono-carboxylic acid salt, in said aqueous polysaccharide composition of step a) to obtain a polysaccharide gel solution, wherein said polysaccharide gel has a concentration of 0.1 to 5.0% by weight of a chitosan or a chitosan derivative, and a concentration of 1.0 to 20% by weight of a salt of a polyol or sugar, and has a pH from about 6.4 to about 7.4.
This method may further comprises a step c) after step b),
c) heating said polysaccharide gel solution at a solidifying temperature ranging from about 20xc2x0 C. to about 80xc2x0 C. until formation of a polysaccharide gel.
A pharmaceutical agent may be added to the polysaccharide gel solution of step b).
The method may further comprises a step i) after step b),
i) dispensing for gelation the polysaccharide gel solution into a desired receiver, either in a mold or within a tissue, organ or body cavity.
The aqueous acidic solution may be prepared from organic or inorganic acids selected from the group consisting of acetic acid, ascorbic acid, salicylic acid, phosphoric acid, hydrochloric acid, propionic acid, formic acid, and a mixture thereof.
The polysaccharide gel solution may be kept in a stable ungelled liquid form at a temperature ranging from about 0xc2x0 C. to about 20xc2x0 C.
The solidifying temperature is ranging from about 37xc2x0 C. to about 60xc2x0 C., preferably about 37xc2x0 C.
The molecular weight of chitosan is ranging from about 10,000 to 2,000,000.
The polysaccharide gel is thermoirreversible or thermoreversible by adjusting the polysaccharide gel pH, when the pH of said polysaccharide gel solution is  greater than 6.9, or when the pH of said polysaccharide gel solution is  less than 6.9.
The solid particulate additives may be added to the polysaccharide gel solution of step b).
The polysaccharide gel solution may be introduced within an animal or human body by injection or endoscopic administration, and gelled in situ at a temperature of about 37xc2x0 C.
In accordance with the present invention there is also provided the use of the polysaccharide gel for producing biocompatible degradable materials used in cosmetics, pharmacology, medicine and/or surgery.
The gel may be incorporated as a whole, or as a component, into implantable devices or implants for repair, reconstruction and/or replacement of tissues and/or organs, either in animals or humans.
The gel may be used as a whole, or as a component of, implantable, transdermal or dermatological drug delivery systems.
The gel may be used as a whole, or as a component of, opthalmological implants or drug delivery systems.
The gel may be used for producing cells-loaded artificial matrices that are applied to the engineering and culture of bioengineered hybrid materials and tissue equivalents.
The loaded cells may be selected from the group consisting of chondrocytes (articular cartilage), fibrochondrocytes (meniscus), ligament fibroblasts (ligament), skin fibroblasts (skin), tenocytes (tendons), myofibroblasts (muscle), mesenchymal stem cells and keratinocytes (skin).
The cells-loaded gel and derived products are devoted to the culture and engineering of artificial articular cartilage and cartilaginous tissues and organs, either for surgical or laboratory testing applications.
The cells-loaded gel and derived products are devoted to the processing and engineering of living artificial substitutes for ligaments, tendons, skin, bone muscles and any metabolic organs, either for surgical or laboratory testing applications.
The cells-loaded gel and derived products are applied as living substitutes for the replacement of articular cartilages, fibrocartilages, cartilaginous organs, ligaments, tendons, bone tissues or skin.
The cells-loaded hydrogel is gelated in situ to induce an ectopic formation of fibrocartilage-like or cartilage-like tissues.
In accordance with the present invention there is also provided the use of loaded polysaccharide gel as injectable or implantable gel biomaterials which act as supports, carriers, reconstructive devices or substitutes for the formation in situ of bone-like, fibrocartilage-like or cartilage-like tissues at a physiological location of an animal or a human.
The polysaccharide gel solution may be used for producing a derived gel or hydrogel by 1) incorporating and dissolving at least one complementary polymer within said polysaccharide gel solution, and 2) by allowing said polysaccharide and complementary polymer to interact for a sufficient period of time to turn into a clear three-dimensional gel within a temperature range between 20xc2x0 C. to 60xc2x0 C.
The complementary polymer is a non-ionic water-soluble polysaccharide or a hydroxyalkyl cellulose.
For the purpose of the present invention the following terms and expressions are defined below.
The term xe2x80x9cpolysaccharide gel solutionxe2x80x9d is intended to mean a polysaccharide solution in a stable ungelled liquid form at a temperature ranging from about 0xc2x0 C. to about 15xc2x0 C. which can be gelated or changed to a gel state when heated at the gelating temperature.
The term xe2x80x9cgelating temperaturexe2x80x9d is intended to mean any temperature ranging from about 20xc2x0 C. to about 80xc2x0 C., preferably between 37xc2x0 C. to about 60xc2x0 C., and more preferably at about the physiological temperature or 37xc2x0 C.
The expression xe2x80x9csalts of polyols or sugarsxe2x80x9d is intended to mean mono-phosphate di-basic salts, mono-sulfate salts-and mono-carboxylic acid salts of polyols or sugars.
The present invention include method of forming different gelated materials, those materials being either molded (customized shapes, tubes, membranes, films . . . ) or formed in situ within biological environments (filling of tissue defects).
In a preferred embodiment, the chitosan/organo-phosphate aqueous solution has a pH above the pKa of chitosan and turn into solid gel upon thermal stimulation. This polysaccharide gel can be used as a carrier for drugs or as a non-living therapeutics delivery system, as substituting materials for tissues and organs and as encapsulants for living cells or microorganisms. Chitosan/organo-phosphate gel matrices are rapidly formed at temperatures between 30 to 60xc2x0 C. Chitosan/organo-phosphate aqueous systems are used as injectable filling materials, injected and gelated in situ for filling and repairing tissue defects.
Glycerol-2-phosphate, glycerol-3-phosphate and glucose-1-phosphate based salts are the preferred disclosed salts in accordance with the present invention.
Chitosan/polyol- or sugar-phosphate and chitosan/polyol- or sugar-sulfate gels can be applied to surgical reconstructive and regeneration uses and drug delivery purposes. They provide thermally reversible or irreversible bioerodible polymeric gels with biologically well-known and compatible components for a broad range of medical/biotechnological applications.